Probiotics and prebiotics are CAM interventions frequently employed by the American public to promote intestinal health and wellness. The presence of beneficial bacteria such as lactobacilli and bifidobacteria, whether delivered exogenously as probiotics or enriched via prebiotics, has been linked to positive health effects including reduction of gut inflammation, diarrhea and allergic reactions. At present however, our understanding of the mechanism of action underlying these biological effects is significantly lacking. Milk oligosaccharides are naturally-evolved prebiotic substrates that facilitate bifidobacterial enrichment and interaction within the infant host. Work from the UC Davis Milk Bioactives Program has shown that human milk oligosaccharides are utilized by select infant-borne bifidobacterial strains that demonstrated unique preferences in oligosaccharide consumption. In addition, genomic analysis of Bifidobacterium infantis and Bifidobacterium bifidum, two strains that grow well on milk oligosaccharides, has revealed unique gene clusters that are specifically induced during growth on these glycans. Further analysis has revealed that growth on milk glycans results in enhanced bifidobacterial interaction with the host epithelium. We hypothesize that the evolutionarily-driven relationship between milk oligosaccharides and cognate infant-borne bifidobacteria provides a model for enhanced probiotic persistence within, and interaction with, the human host. To address this hypothesis we will gain mechanistic insight into the specific catabolism of these unique milk glycans by bifidobacteria and comprehensively map their influence on bifidobacteria-host interaction via the following Specific Aims: 1. To comprehensively characterize human and bovine milk oligosaccharides and develop methods for their large scale fractionation and production for functional studies. 2. To completely characterize the bifidobacterial transporters and glycosyl hydrolases necessary to deconstruct these complex milk oligosaccharides. 3. To examine the influence of milk oligosaccharides on the interaction between bifidobacteria and the host. Successful completion of these Specific Aims will reveal specific mechanisms by which human milk oligosaccharides facilitate a protective enrichment of bifidobacteria in the gastrointestinal tract of breast fed infants. In addition, we will translate these findings to structurally and functionally equivalent bovine milk oligosaccharides, a commercially accessible substrate that can be easily delivered into CAM foods and therapies aimed at gut health. The significance of this application is a greater mechanistic understanding of the beneficial effects of synbiotic (milk oligosaccahrides plus cognate bifidobacteria) applications thereby providing both strategies and diagnostic measures to better address a variety of disorders marked by intestinal dysbiosis.